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Chatterjee, Jonathan Louis Kaplan, Edouard Evangelisti, Hiroki Takagi,
Varodom Charoensawan, David Rengel, et al.
To cite this version:
Aleksandr Gavrin, Thomas Rey, Thomas Torode, Justine Toulotte, Abhishek Chatterjee, et al..
De-velopmental Modulation of Root Cell Wall Architecture Confers Resistance to an Oomycete Pathogen.
Current Biology - CB, Elsevier, 2020, 30, pp.1-12. �10.1016/j.cub.2020.08.011�. �hal-02955235�
Pathogen
Graphical Abstract
Highlights
d
The SCAR protein API controls actin and endomembrane
trafficking dynamics
d
SCAR proteins of several plant species can support
symbiosis and pathogen infection
d
A mutation in
API affects specific biochemical properties of
plant cell walls
d
An altered wall architecture results in root resistance to
Phytophthora palmivora
Authors
Aleksandr Gavrin, Thomas Rey,
Thomas A. Torode, ..., Ryohei Terauchi,
Siobhan Braybrook,
Sebastian Schornack
Correspondence
sebastian.schornack@slcu.cam.ac.uk
In Brief
Subtly altered plant cell walls can be
decisive for disease. Gavrin et al. show
that the Medicago SCAR/WAVE complex
protein API controls actin cytoskeleton
dynamics in roots. Local changes
associated with a loss of
API establish a
cell wall architecture with altered
biochemical properties that hinder
infection progress by an oomycete
pathogen.
Gavrin et al., 2020, Current Biology30, 1–12
November 2, 2020ª 2020 The Authors. Published by Elsevier Inc.
Article
Developmental Modulation of Root
Cell Wall Architecture Confers
Resistance to an Oomycete Pathogen
Aleksandr Gavrin,1Thomas Rey,1,10Thomas A. Torode,1,10Justine Toulotte,1Abhishek Chatterjee,1 Jonathan Louis Kaplan,1Edouard Evangelisti,1Hiroki Takagi,2Varodom Charoensawan,1,7David Rengel,3,8
Etienne-Pascal Journet,3,9Frederic Debelle,3Fernanda de Carvalho-Niebel,3Ryohei Terauchi,2Siobhan Braybrook,1,4,5,6 and Sebastian Schornack1,11,*
1Sainsbury Laboratory (SLCU), University of Cambridge, 47 Bateman Street, Cambridge CB2 1LR, UK 2Iwate Biotechnology Institute, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan
3LIPM, Universite de Toulouse, INRA, CNRS, Castanet-Tolosan 31326, France
4Department of Molecular, Cell, and Developmental Biology, 610 Charles E Young Drive South, University of California, Los Angeles, Los
Angeles, CA 90095, USA
5Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA
6Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA
90095, USA
7Department of Biochemistry, Faculty of Science, and Integrative Computational BioScience (ICBS) Center, Mahidol University, Bangkok
10400, Thailand
8GeT-PlaGe, Genotoul, INRA US1426, Castanet-Tolosan Cedex, France 9AGIR, Universite de Toulouse, INRA, ENSFEA, Castanet-Tolosan 31326, France 10These authors contributed equally
11Lead Contact
*Correspondence:sebastian.schornack@slcu.cam.ac.uk https://doi.org/10.1016/j.cub.2020.08.011
SUMMARY
The cell wall is the primary interface between plant cells and their immediate environment and must balance
multiple functionalities, including the regulation of growth, the entry of beneficial microbes, and protection
against pathogens. Here, we demonstrate how API, a SCAR2 protein component of the SCAR/WAVE
com-plex, controls the root cell wall architecture important for pathogenic oomycete and symbiotic bacterial
in-teractions in legumes. A mutation in
API results in root resistance to the pathogen Phytophthora palmivora
and colonization defects by symbiotic rhizobia. Although
api mutant plants do not exhibit significant overall
growth and development defects, their root cells display delayed actin and endomembrane trafficking
dy-namics and selectively secrete less of the cell wall polysaccharide xyloglucan. Changes associated with a
loss of
API establish a cell wall architecture with altered biochemical properties that hinder P. palmivora
infection progress. Thus, developmental stage-dependent modifications of the cell wall, driven by SCAR/
WAVE, are important in balancing cell wall developmental functions and microbial invasion.
INTRODUCTION
The cell wall protects plant cells from microbial invasion while maintaining properties enabling growth and development. Cell wall structures and modifications, such as the waxy cuticle and lignification, provide mechanical barriers for entry attempts [1]. Additionally, reinforcement of the cell wall through the depo-sition of carbohydrates results in the formation of papilla struc-tures at attempted penetration sites [2]. Despite these measures, adapted pathogens have evolved strategies to ensure their pas-sage through the cell wall. This raises the important question to what extent selective plant cell wall architecture changes can result in pathogen resistance while maintaining other biologically important properties, such as normal growth [3].
Phytophthora palmivora belongs to a genus of aggressive
hemibiotrophic pathogens causing diseases in many important
tropical crops [4]. P. palmivora has a wide host spectrum and is able to infect root and leaf tissues of several plant species, ranging from liverworts [5] to monocotyledonous flowering plants [6] and including legumes widely used in symbiosis research [7]. During root infection, mobile P. palmivora zoo-spores accumulate just above the root cap [8], where they encyst and form germ tubes with terminal appressoria to penetrate the subapical root epidermis and rapidly colonize the root cortex. Entry is facilitated in part through localized secretion of plant-cell-wall-degrading enzymes [9]. In the cortex, P. palmivora grows mostly intercellularly and projects short specialized hy-phae, termed haustoria, through the walls of individual living root cells, resulting in the invagination of their protoplast. This is followed by a necrotrophic stage, characterized by host tissue necrosis and the formation of sporangia, which release new zoo-spores for further infection [10]. Unlike pathogenic interactions
where cell wall modifications may block microbial entry, symbi-otic interactions rely on cell wall remodeling to guide microbial entry and facilitate the establishment of nutrient exchange inter-faces [11]. Rhizobia infection of roots of model legumes, such as
Medicago truncatula and Lotus japonicus, occurs via host-driven
tubular tip-growing structures known as infection threads (ITs). These tubular structures are initiated within curled root hairs following a two-step process, which involves localized host cell wall remodeling before IT polar tip growth initiates and pro-gresses along the root hair length [12]. Mutational analyses in
M. truncatula and L. japonicus have revealed that targeted
secre-tion of cell wall polysaccharides, local degradasecre-tion of plant cell walls, and cytoskeleton rearrangements are required for normal initiation and progression of ITs [13–19].
Plant cell wall biosynthesis relies on cellular secretory pro-cesses and the cytoskeleton. Major structural components of the primary walls are cellulose, hemicelluloses, and pectins. The polysaccharides, remodeling proteins, and some biosyn-thetic machinery that generate the cell walls are delivered through endomembrane trafficking [20,21]. Cellulose is synthe-sized at the plasma membrane by a membrane-deployed cellu-lose synthase complex, whereas hemicellucellu-loses and pectins are synthesized in the Golgi by sequential modification of the side chains in the various Golgi cisternae. The transport of synthe-sized polysaccharides to the plasma membrane is generally considered to be mediated by the default exocytosis pathway [22]. Xyloglucan is the most abundant hemicellulose in the pri-mary cell wall of angiosperms, where it binds to cellulose micro-fibers and may link them together [23]. Furthermore, xyloglucan is secreted by root cells into the surrounding soil, where it acts as an efficient soil aggregator [24].
Deposition of pectins and hemicelluloses at the cell wall is largely dependent on the dynamics of the actin cytoskeleton, which at a whole-cell spatial scale dictates the patterns of intra-cellular transport and secretion [22]. Therefore, actin filaments directly affect plant cell wall establishment and remodeling. The formation and maintenance of the actin network is largely dependent on actin-binding proteins and actin filament nuclea-tors. In plants, formins and ARP2/3 (actin-related protein 2/3) are two important actin filament nucleators, which confer actin filament branching. The ARP2/3 complex, in its active conforma-tion, nucleates actin filaments from the sides of an existing fila-ment and initiates a daughter filafila-ment at a 70angle [25]. Plant ARP2/3 activation relies solely on the heteromeric five-subunit SCAR/WAVE (suppressor of cyclic AMP [cAMP] Receptor/Wis-kott-Aldrich syndrome protein-family Verprolin homologous pro-tein) regulatory complex [26]. The SCAR/WAVE complex contrib-utes to diverse processes and aspects of plant development, including asymmetric cell divisions and cell morphology [27, 28], trichome architecture, plant cell adhesive properties [29], root rigidity, cell-cell junction [30], and rhizobial infection thread progression [15,16,18].
SCAR proteins constitute core elements of the SCAR/WAVE complex. Their N-terminal SCAR homology domain (SHD) ap-pears to regulate protein stability and their assembly into the SCAR/WAVE complex [31], although their C-terminal WCA/ VCA (WA) domain is sufficient for ARP2/3 activation [32].
Arabi-dopsis encodes four SCARs, each with a similar domain
organi-zation [33]. Mutations in AtSCAR2 are sufficient to cause a mild
distorted trichome phenotype [29,34]. The overlapping expres-sion patterns of the four Arabidopsis SCAR genes and the rela-tively weak phenotypes of scar2 mutant lines suggest potential functional redundancy among respective family members [35]. Although a role for SCAR/WAVE proteins has been reported in the context of plant symbiosis [16] and in pathogenic interactions in animals [36], their importance for plant-pathogen interactions has not been addressed.
To identify genetic components commonly required for the ac-commodation of pathogenic and symbiotic microbes, we carried out root oomycete infection assays on M. truncatula mutant seedlings affected in rhizobia-root colonization. We demonstrate that the Medicago API gene, as well as its L. japonicus and
Ara-bidopsis orthologs, can control cell wall properties required for
efficient Medicago root entry by the oomycete pathogen
P. palmivora. API encodes a SCAR2 protein, a subunit of the
plant SCAR/WAVE actin regulatory complex. Quantitative resis-tance to P. palmivora is likely attributable to a modified plant cell wall architecture, rather than an altered defense response tran-scriptome. We demonstrated impaired actin and endomem-brane trafficking dynamics in api mutants, resulting in the distor-tion of secreted cell wall remodeling factors. This leads to changes in biochemical properties of root cell walls that likely impair pathogen root entry without affecting overall plant growth. Our work demonstrates that alterations in the cell wall architec-ture of specific root cells contribute to disease resistance during a compatible interaction without compromising root growth, of-fering a potential route to quantitative root resistance against
Phytophthora.
RESULTS
Plants Mutated in the SCAR2 Gene API Are
Compromised in Root Entry of a Pathogenic Oomycete
When surveying mutants impaired in symbiosis [10] for their abil-ity to resist pathogen infection, we found that seedling roots car-rying the api (altered nodule primordia invasion) mutation [37] displayed significantly reduced disease symptoms upon infec-tion with P. palmivora zoospores. Visual symptoms, pathogen biomass, and defense gene activation were reduced in the api mutant (Figures 1A–1C). Microscopic inspection revealed that
P. palmivora infections were hindered at the root entry stage
and infectious intraradical hyphae were shorter and less frequent in api compared to wild type (Figures 1D–1G). Reduced penetra-tion was not isolate specific and was consistently observed with both the P. palmivora AJ-td strain from Indonesia as well as with LILI-td originating from Colombia, which also formed haustoria in api mutants (Figure S1A). At 4 days post inoculation (dpi), P. palmivora LILI-td produced significantly less sporangia on api mutants compared to wild type (Figures 1H and 1I). Taken together, these results indicate that the api mutant is compro-mised in P. palmivora colonization and sporulation.
The api mutant was initially reported as rhizobia-infection defective, resulting in the frequent development of non-invaded underdeveloped nodule primordia with large infection pockets and a reduced root hair length phenotype [37]. We confirmed the rhizobia-infection defective phenotype of api using fluores-cently labeled Sinorhizobium meliloti bacteria. Overall, in our conditions, we found that 70% of api nodules are non-invaded
outgrowths where bacteria accumulated in micro-colonies be-tween cells of the root nodule primordia (Figure 2A).
To identify the genetic basis of the observed cell entry pheno-types, we combined Illumina sequencing of DNA from wild-type and mutant genotypes with transcriptome expression analyses (Data S1A). The previously genetically mapped target interval [37] was further narrowed down (Figure S1B). We identified a G2324A mutation in the SCAR2 protein-encoding gene Mtru-nA17_Chr4g0004861 (Medtr4g013235) that results in an early stop codon (Figure S1C). Transcript levels of Mtru-nA17_Chr4g0004861were attenuated in the mutant in both Affy-metrix microarray data and quantitative reverse transcriptase PCR (qRT-PCR) analysis (Data S1A;Figure S2D). The gene en-coded by MtrunA17_Chr4g0004861 was termed API in accor-dance with the previously described genetic locus [37]. In SCAR/WAVE complex mutants, leaf trichomes and overall development are frequently altered [15, 29,38]. By contrast, we did not observe any changes in overall development, seed production, or trichomes between wild-type and api mutants be-sides the reduced root hair length (Figures S1E–S1H), and previ-ously reported leaf chlorosis and reduced root growth were attributed to a reduced nitrogen fixing ability [37]. Microscopic investigation of sectioned and toluidine-blue-stained roots sug-gests that api mutants overall display a normal cellular architec-ture. In only 4 of 30 investigated roots, we observed small groups of cells with altered organization, which, however, did not impact on overall root development (Figures S1I–S1P).
Expression of the API gene in transgenic roots of api under Ubiquitin 3 (Ub3) promoter or a 3.5-kb upstream sequence restored root hair length, wild-type frequencies of bacterial-colo-nized nodules, and full susceptibility to P. palmivora root
Figure 1. TheM. truncatula api Mutant Is Resistant to Root Cellular Entry of the Pathogenic OomyceteP. palmivora
(A) Visual disease symptoms of API (wild-type) and api seedlings after root tip infection with P. palmivora AJ-td zoospores at 72 h post-inoculation (hpi). Red arrowheads mark the extent of infection symptoms toward hypocotyl (green arrowhead); scale bars, 1 cm.
(B) Scoring of visual disease symptom extent (symptom length/seedling length) of API (n = 58) and api (n = 48) plants inoculated with P. palmivora AJ-td at 72 hpi (error bars represent SEM; t test: ***p < 0.001).
(C) Quantification of the plant immunity marker MtGERMIN and P. palmivora biomass marker PpEf1a at 16 hpi by qRT-PCR using the 2DCpmethod and MtH3l as a reference gene (error bars represent SEM; n = 3, t test: *p < 0.05). (D) Epifluorescence microscopy of API and api seedlings at 12 hpi with P. palmivora AJ-td. Arrowheads indicate infections with intraradical growing hyphae. Asterisks indicate infections limited to the epidermis cell; scale bars, 100mm. See alsoFigure S1A.
(E) Average length of all P. palmivora AJ-td intraradical hyphae per seedling at 12 hpi (seedlings analyzed: n = 10 per genotype; error bars represent SD; t test: **p < 0.01).
(F) Total amount of infections at the stage of the epidermis cell penetration per seedling at 12 hpi (seedlings analyzed: n = 10 per genotype; error bars represent SD; t test: **p < 0.01).
(G) Total amount of infections with intraradical hyphae per seedling at 12 hpi (seedlings analyzed: n = 10 per genotype; error bars represent SD; t test: **p < 0.01).
(H) Epifluorescence microscopy of API and api seedlings at 4 dpi with P. palmivora LILI-td. Scale bars, 150mm.
(I) Total amount of sporangia per seedling at 4 dpi (seedlings analyzed: n = 25 per genotype; error bars represent SD; t test: ***p < 0.001).
infection as measured by pathogen biomass, respectively ( Fig-ures S2A–S2I). To obtain independent evidence for the role of
API in P. palmivora infection, we used a hairpin RNAi construct
targeting the 30 UTR of MtrunA17_Chr4g0004861 (hpAPI) to attenuate API transcript levels in wild-type roots. Roots express-ing the hpAPI construct displayed the shorter root hair pheno-type (Figures S2J and S2K), in contrast to control roots express-ing a construct targetexpress-ing the non-existent uidA gene (hpuidA). We further confirmed that hpAPI-expressing transgenic roots in-fected with P. palmivora showed a significant reduction in API transcripts concomitantly with a reduction of P. palmivora biomass markers (Figure S2L). Taken together, the data demon-strate that the SCAR2 protein API is required for efficient coloni-zation of M. truncatula by the filamentous pathogen P. palmivora and symbiotic rhizobia and api plants do not display altered overall plant development.
SCAR2 Functionality Is Maintained in Different Plant Species
API belongs to a small family of SCAR-related proteins found across plant species (Figure S3A). The L. japonicus API ortholog
LjSCARN has been proposed to function in nodulation [16]. However, when expressed under ubiquitin- or epidermis-spe-cific EXPA7 promoter [39] in roots of composite plants, SCARN can complement all M. truncatula api mutant root phenotypes (Figures 2B,S3B, and S3C), suggesting that SCARN’s function-ality is not restricted to root nodulation but is also involved in an oomycete pathogen interaction. In support, L. japonicus scarn-1 mutants were also more resistant to P. palmivora root infection (Figure 2C). Furthermore, ubiquitin-promoter-driven expression of AtSCAR2, the closest homolog of API from Arabidopsis, was also able to complement all api mutant phenotypes (Figures 2B, 2D,S3D, and S3E). Therefore, SCAR2-related proteins of different plant species can support rhizobia and P. palmivora infection in M. truncatula as well as in L. japonicus.
API and Its Homologs Are More Strongly Expressed in Root Meristems and Nodule Primordia Tissues
We investigated the expression patterns of API and its close ho-mologs HAPI1 (homolog of API 1) and HAPI2 in transgenic roots of Medicago composite plants by examining the activity of tran-scriptional 2-kb-promoter fusions to GUS (b-glucuronidase) or a nuclear localized mTFP (monomeric teal fluorescent protein) and by employing qRT-PCR. GUS activity was mainly detectable in the meristematic root tip region, in root hairs, and in dividing cells of nodule primordia (Figures 3A and 3F). The mTFP reporter confirmed this expression in the root tips and nodule primordia (Figures 3C and 3E) as well as in root epidermal cells and root hairs (Figures 3B and 3D). At that stage, we could not detect significantly elevated levels of GUS activity associated with infection thread progression or at sites of P. palmivora infection. Instead, GUS and mTFP signals appeared weaker in pathogen-infected roots (Figures 3E and 3F). However, qRT-PCR did not show a statistically significant reduction. API transcript levels re-mained unaltered during P. palmivora infection (Figure 3G) but increased over time during nodule primordia development ( Fig-ure 3H). HAPI1 and HAPI2 showed similar expression patterns in infected and non-infected roots as well as in young developing nodule primordia (Figures 3G, 3H, andS4). In the api mutant
Figure 2. SCAR2-Related Proteins of Different Plant Species Can Support Rhizobia andP. palmivora Infection
(A) Microscopy of API and api nodules at 18 days post-inoculation (dpi) with GFP-expressing Sinorhizobium meliloti 2011 (plants analyzed: n = 20 per ge-notype; scale bars, 150mm).
(B) Epifluorescence microscopy of root nodules in api hairy roots expressing Ub10:dsRed, Ub:LjSCARN, and Ub3:AtSCAR2. Asterisks indicate fully developed bacteria-filled nodules at 18 dpi; arrowheads indicate non-invaded outgrowths with arrested infection foci on top (green fluorescence); scale bars, 1 mm. See alsoFigure S3D.
(C) Quantification of the P. palmivora AJ-td biomass marker PpEf1a in wild-type L. japonicus Gifu and scarn-1 mutant by qRT-PCR using the 2DCp method and LjUBQ as a reference gene (error bars represent SD; biological replicates n = 3; t test: *p < 0.05).
(D) Quantification of the P. palmivora LILI-YKDel biomass marker PpEf1a in API and api transgenic roots expressing 35S:GFP (controls) and api roots expressing Ub3:AtSCAR2 by qRT-PCR using the 2DCpmethod and MtUBQ as a reference gene (error bars represent SD; biological replicates n = 5; t test: *p < 0.05). See alsoFigures S2andS3E.
background, we did not detect compensating higher transcript levels of HAPI1 and HAPI2 (Figure 3G). Together, these data sug-gest that Medicago SCAR genes are not induced upon early
P. palmivora infection and their upregulation during nodule
development is likely attributable to cell division activity of nodule primordia rather than linked to infection thread progres-sion in the cortex.
Induced Responses to Pathogen Infection Are Unaltered in api Mutants despite Impaired Actin Dynamics
We next addressed whether api mutants display an altered tran-scriptional response to P. palmivora infection. Microarray experi-ments indicated only minor changes in transcript levels between
uninfected wild-type and mutant seedlings: just 44 of the 50,900 probes hybridized differentially to 32 assignable transcripts (log2FC > 1;Data S1A;Figure S5A). This indicates that the api mu-tation does not lead to massive constitutive deregulation of the transcriptome. Just a single probe matching a putative transcript of the Mapman biotic stress category [40] was differentially ex-pressed with a log2FC = 1.05, illustrating an absence of a consti-tutively heightened defense response. Infection by P. palmivora AJ-td deregulated a more significant set of transcripts (Data S1B). However, the infection resulted in similar overall transcrip-tome dynamics when comparing wild-type and api seedlings ( Fig-ure S5B) with just 65 differential probes between both genotypes, four of them assigned to ‘‘biotic stress’’ genes (Data S1C).
Figure 3. SCAR Genes Are Preferentially Expressed in Root Meristems and Nodule Primordia
(A) GUS staining of uninfected transgenic Medicago roots expressing a pAPI:GUS reporter.
(B–D) Expression of a pAPI:NLS:mTFP reporter in epidermal cells of the maturation zone (B), meristem and elongation zone (C), and a root hair cell (D). (E) Medicago roots expressing a pAPI:NLS:mTFP reporter at mock, 24 h upon P. palmivora LILI-YKDel infection, and 4 days post-inoculation with GFP expressing S. meliloti. Open arrowhead indicates dividing cells of a nodule primordium; closed arrowhead indicates an infection thread.
(F) GUS staining of transgenic Medicago roots expressing pAPI:GUS reporter at mock, 24 h upon P. palmivora LILI-YKDel infection, and 4 days post-inoculation with GFP expressing S. meliloti. Open arrowhead indicates dividing cells of a nodule primordia; closed arrowhead indicates an infection thread.
(G) Transcript levels of API, HAPI1, and HAPI2 genes upon P. palmivora AJ-td inoculation in API and api background. qRT-PCR was analyzed using 2DCpmethod and MtUBQ as a reference gene (error bars represent SD; biological replicates n = 3; t test: *p < 0.05).
(H) Transcript levels of API, HAPI1, and HAPI2 genes at early stages of root nodule development in wild-type background. qRT-PCR was analyzed using 2DCp method and MtUBQ as a reference gene (error bars represent SD; biological replicates n = 3; one-way ANOVA with post hoc Tukey HSD test p < 0.05). See alsoFigures S4andS5andData S1andS2.
Overall similar microarray gene expression profiles were further confirmed by qRT-PCR analysis, and similar levels of pre-viously published pathogen-induced transcripts PRX, GLP, LOX, and THA [41,42] were quantified in both mutant and wild-type contexts (Figure S5C;Data S2).
Because SCAR2 proteins have been reported to regulate actin processes [35], we assessed whether actin responses were affected in the api mutant background upon microbial infection. Confocal fluorescence microscopy of wild-type and api mutant roots expressing the ABD2-YFP (actin binding domain2-yellow fluorescent protein) actin reporter did not reveal changes in den-sity and bundling of actin in cortical cells of root areas targeted by P. palmivora (Figures S5D–S5F). P. palmivora penetration sites of api and wild-type seedlings showed similar levels of closely positioned plant nuclei (Figure S5G), indicating no signif-icant impairment of actin-mediated nuclear repositioning in api. Furthermore, no difference in actin bundle distribution was observed in developing root nodules of both wild-type and mutant plants (Figure S5H).
However, api mutants are quantitatively impaired in actin dy-namics in epidermal and cortical cells of the root elongation zone (Figure 4). This is revealed by consecutive time-lapse im-ages showing a higher correlation coefficient and a lower pixel difference in api mutants than in API wild type (Figures 4A and 4B). Importantly, api mutants still retained the ability for dynamic actin changes compared to roots treated with the inhibitor MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), and actin dynamics upon P. palmivora infection are not different be-tween api and wild type (Figure 4B). Taken together, although
transcriptomic changes associated to defense responses were similar in wild-type and api roots, api mutants display impaired actin dynamics but remain unaltered in actin network density and bundling.
API Controls Endomembrane Trafficking to Establish Cell Wall Properties Compatible with Infection Processes
To investigate whether altered actin dynamics in api result in quantitative differences in secretory processes, we monitored how fast the plasma membrane integral aquaporin PIP1-GFP fusion is redeployed after photobleaching (Figure 5A). The recov-ery of PIP1-GFP fluorescence after photobleaching was signifi-cantly reduced in cells of division and elongation zones of api roots, but no difference was observed in generally slower recov-ering mature root cells (Figures 5B–5E). This suggests that altered endomembrane dynamics are restricted to those root tis-sues that display pronounced activity. We subsequently moni-tored brefeldin-A (BFA)-induced BFA-body formation in the outer cortex of api root elongation zones over time using a GFP secre-tion reporter system [43]. These analyses revealed that accumu-lation of GFP-labeled endomembrane compartments into a BFA body was delayed in api, resulting in the formation of small BFA bodies (Figures S6A and S6B). This delay was not caused by dif-ferences in general chemical permeability between wild-type and api mutants (Figure S6A).
We then tested whether the delayed endomembrane dy-namics in api may affect the secretion of specific cell wall com-ponents. An immunoblot of root secretions probed with
Figure 4. The Elongation Zone ofapi Roots Displays Impaired Actin Dynamics
(A) Representative images from a single focal plane time-lapse series at 2.5-s intervals of API and api hairy root epidermal cells expressing YFP-ABD2 actin reporter. The merged image (far-right column) shows all three time points as separate color channels in a red, green, and blue (RGB) image. MBS-treated cells exhibit very minor changes in actin filament organization over the time course analyzed, and this is represented by an almost completely white overlay in the merged image. Scale bars represent 3mm.
(B) Quantification of actin dynamics in API and api background upon infection with P. palmivora and mock conditions using two independent methods: total differences per pixel and correlation coefficient (ten transgenic roots were analyzed for each condition; cells analyzed: mock nAPI= 27, napi= 26; infected nAPI= 23,
napi= 41; error bars represent SE; two-way ANOVA with post hoc Tukey HSD test p < 0.05).
monoclonal antibodies (mAbs) toward specific cell wall compo-nents indicated a reduction in secreted xyloglucans in api mu-tants relative to wild-type API controls, as detected by the xylo-glucan-specific antibody LM25 (Figures 5F and 5G). In contrast, no differences were observed when using antibodies toward pectic polysaccharides of homogalacturonan (via LM19) or RG-I (via LM5, LM26, and LM6-M;Figure S6C). These results suggest a role for SCAR2 proteins in cell wall xyloglucan secre-tion. Nevertheless, the external application of xyloglucans to api seedlings did not restore susceptibility to P. palmivora ( Fig-ure 5H). Taken together, api roots display delayed endomem-brane compartment trafficking and altered selective secretion of root cell wall components.
We next addressed whether api root cell walls exhibited altered polysaccharide composition. Wall extracts from three developmental zones of seedling roots (regions I to III inFigure 6) were analyzed via enzyme-linked immunosorbent assay (ELISA) using a range of mAbs toward cell wall polysaccharides (see STAR Methods). No alteration in composition or abundance of any of the tested cell wall components was detected within frac-tions extracted with calcium chelating (CDTA) and alkaline buffers (KOH), and in support, immunolocalization of different epitopes in cross sections did not reveal any conspicuous differences (Figures 6 and S7). However, solubilization of the cellulose microfibrils and their tightly associated polysaccha-rides as a cellulose-associated fraction (CAF) [44] revealed an
Figure 5. api Mutants Display Delayed PIP1 Plasma Membrane Protein Recovery and Reduced Secretion of Xyloglucan
(A) Fluorescence recovery after photobleaching within 40 min measured in inner root cells of elongation and maturation zones of API and api transgenic roots expressing Ub3:PIP1-GFP.
(B and C) Fluorescence recovery kinetics of PIP1-GFP in API (B) and api (C) transgenic roots (API: nroot tip= 11, nmature root= 11; R2root tip= 0.95, R2mature root= 0.97;
api: nroot tip= 12, nmature root= 10; R
2
root tip= 0.95, R
2
mature root= 0.99).
(D) Quantification of the mobile fraction contributing to fluorescence recovery of PIP1-GFP at the membrane measured in different root areas of API and api transgenic roots expressing Ub3:PIP1-GFP (error bars represent SE; number of FRAP: root tip nAPI= 11, napi= 12; mature root nAPI= 11, napi= 10).
(E) Quantification of the halftime of recovery in different root areas of API and api transgenic roots expressing Ub3:PIP1-GFP (error bars represent SE; number of FRAP root tip nAPI= 11, napi= 12; mature root nAPI= 11, napi= 10; t test: *p < 0.05).
(F) Immunodetection of xyloglucan (LM25) secretion from plant surfaces. Bright-field image of API and api seedlings grown on agar solid media superimposed with the nitrocellulose print of the solid media surface after removal of seedlings is shown, which was then probed with LM25 antibodies (n = 10 per genotype). See alsoFigure S6C.
(G) Quantification of LM25 antibody signal (error bars represent SD; biological replicates n = 4 per genotype; t test: ***p < 0.001).
(H) Quantification of the P. palmivora biomass in xyloglucan-treated infected seedlings by qRT-PCR using the 2DCpmethod and MtUBQ as a reference gene (error bars represent SE; biological replicates n = 3; one-way ANOVA with post hoc Tukey HSD test p < 0.05).
api-specific reduction of epitopes recognized by mAbs toward
xyloglucans (LM15 and LM25), RG-I (LM5 and LM26), and homo-galacturonan (LM19) in the specific zones I and II comprising meristematic, elongation, and root hair differentiation zones of the root (Figure 6). This transient decrease in detectable cellu-lose-associated pectin and xyloglucan in the api mutants sug-gests an alteration in the cell wall architecture in root zones where the preferential colonization by P. palmivora takes place. Our data collectively revealed significant differences in actin dynamics, endomembrane secretion processes, cell wall biochemistry, and architecture in api mutants relative to wild-type Medicago plants. To specifically assess the contribution of altered cell wall properties rather than actin dynamics, endo-membrane trafficking, and secretion processes to the pathogen phenotype, we infected seedlings that were chemically inacti-vated using chloroform treatment. At 20 h post-application of spores, P. palmivora hyphae colonizing these dead seedlings were shorter in api mutants compared to wild type (Figure 7). Thus, P. palmivora colonization of api root tissues is impaired even if these tissues are dead. This suggests that API mediates establishment of a preformed cell wall architecture that supports full seedling colonization by this oomycete. By contrast, api mu-tants present a subtly altered cell wall architecture that is quan-titatively more resistant.
DISCUSSION
We have identified a premature stop codon mutation in the SCAR2-protein-encoding gene API as being responsible for increased quantitative resistance to P. palmivora root infection through the modulation of cell wall properties relevant for infection. A L. japonicus SCARN gene mutation has previously been found to alter root hair length and to impair rhizobia infec-tion during root nodule symbiosis in L. japonicus. On the basis of phylogenetic analyses, the authors had suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis [16]. We demon-strate here that SCAR2-mediated phenotypes in M. truncatula
Figure 6. Cell Walls of api Display Altered Biochemical Properties
ELISA analysis of cell wall polysaccharides ex-tracted sequentially with CDTA, KOH, and cellulase treatment yielding the cellulose-associated fraction (CAF) from three root developmental zones: comprising (I) the root meristem and elongation zone; (II) the root hair differentiation zone; and (III) the mature root zone of API and api roots (error bars represent SD; biological replicates n = 3; t test: *p < 0.05). See alsoFigure S7.
and L. japonicus are not limited to nitrogen fixing symbiosis but also impact root susceptibility to an oomycete pathogen. Therefore, API constitutes a gene commonly contributing to symbiotic and pathogenic microbe colonization in different plants. Furthermore, we showed that ectopic expression of Arabidopsis
SCAR2 was able to complement all observed api mutant
pheno-types, suggesting that legumes have not evolved additional functionality for SCAR2 proteins during rhizobial colonization.
Notably, mutation of RIT (required for infection thread), encod-ing the core SCAR/WAVE complex component NAP1 (Nck-Associated Protein 1), results in an arrest of rhizobia infection threads at earlier stages of colonization and visible changes in aboveground trichome development in M. truncatula [15]. By contrast, api mutants did not display such dramatic develop-mental defects (Figures S1E and S1F). Although infection threads are partially defective in api, they can still initiate and progress through root hairs [37], even though nodule primordia colonization is strongly impaired. It is tempting to speculate that different members of the SCAR family integrate into different SCAR/WAVE complexes consisting of the other components, including NAP1, and therefore, loss of function of a single
SCAR gene only impairs a subset of cell processes. API is
stronger expressed in root meristems, and PIP1-GFP recovery after photobleaching was faster in these tissues. It is plausible that API may contribute more in tissues with a higher demand in cytoskeleton-mediated trafficking processes.
SCAR/WAVE mutants often display very specific and
restricted phenotypes, such as trichome changes [29] or root hair length (Figure S2F), and higher order Arabidopsis SCAR/ WAVE gene mutants are not massively impaired in all develop-ment processes [35]. Cell morphological changes in api are limited to shorter root hairs and rare events of altered cell groups (Figure S1). It is likely that overall root development is thus main-tained through the activity of remaining API homologs as well as other actin regulators. Future work will aim at elucidating whether and how different SCAR proteins confer specific func-tions in plant-microbe interacfunc-tions as well as growth and devel-opment in specific tissues. An alternative, albeit less likely, pos-sibility for the absence of any dramatic developmental defects in
api could be its residual transcript levels (Figure S1D). Nonethe-less, this would result in the expression of a truncated protein lacking the essential C-terminal domains that confer the link to the ARP2/3 complex.
Previous studies have highlighted the potential role of cell-wall components to support pathogen infection [3], but the underly-ing mechanisms remain to be clarified. We demonstrated that the cellulose-associated fractions of api root meristematic, elon-gation, and differentiation zones display reduced levels of detectable epitopes of xyloglucan (LM15 and LM25), RG-I (LM5 and LM26), and homogalacturonan (LM19). A reduction of these polysaccharides in the cellulose-associated fraction of
api suggests an altered cell wall architecture in these particular
root zones. Reduced tethering of xyloglucan and pectic carbo-hydrates during synthesis of cellulose microfibrils is expected to alter the wall’s rheological behavior [45] and influence path-ogen penetration. The defective incorporation of non-cellulosic polysaccharides into cellulose microfibrils in api is likely caused by changes in cytoskeleton-mediated endomembrane compart-ment dynamics, as visualized through impaired actin dynamics and delayed membrane protein delivery, as well as delayed BFA body formation (Figures 4,5, andS6). This likely impacts on the secretory compartment system for delivering cell wall pectins, xyloglucans, and associated remodeling enzymes. Therefore, our data support a link between API SCAR/WAVE protein-mediated actin cytoskeleton rearrangements, secretory processes, and the extracellular matrix/cell wall composition, ul-timately affecting microbial entry at root elongation and differen-tiation zones.
SCAR genes are expressed constitutively during infection, but
the activity of promoter reporter gene fusions was higher in actively dividing tissues of the root tip and nodule primordia ( Fig-ure 3). These actively dividing tissues have a high demand for cell wall material deployment. It is therefore reasonable that a muta-tion in api has a stronger impact in these tissues. Delayed endo-membrane dynamics in api roots lead to an extended area with altered cell wall properties compared to wild type (Figure 6), reaching into the root hair emergence zone, where rhizobial sym-biotic colonization takes place. Importantly, P. palmivora zoo-spores preferentially accumulate at root elongation zones [43, 46], where api mutants display altered cell wall properties. It is tempting to speculate that microbial infections in mature root sections remain unaltered, contributing to the quantitative nature of the phenotype. Consistently, no significantly altered
arbuscular mycorrhizal fungal symbiosis was observed in api mutants [37]. A benefit of such developmental stage-specific resistance is that plants are resilient to pathogens targeting this specific region, such as P. palmivora, without being impaired in their overall root system, shoot size, and seed production. Re-ported plant resistance strategies against P. palmivora include the expression of pathogenicity-related genes and antimicrobial secondary metabolites [47]. Furthermore, a diversity panel sur-vey in M. truncatula failed to identify completely resistant acces-sions. Instead, polymorphisms near RAD1 were correlated with the extent of root disease symptoms [8]. These works and our study highlight the importance of quantitative disease resistance in the control of this pathogen.
STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Plant materials and microbial strains
d METHOD DETAILS
B Genetic mapping and SNPs analysis
B Design of constructs and cloning
B Microscopy
B Promoter GUS assay
B Quantification of actin dynamics and organization
B FRAP acquisition and analysis
B Endomembrane dynamics analysis
B Cell wall extraction, ELISA and immunolabelling
B Gene expression assays
B Microarray
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online athttps://doi.org/10.1016/j. cub.2020.08.011.
ACKNOWLEDGMENTS
We are indebted to Paul Knox (Leeds) for provision of cell wall antibodies and to Jean-Marie Prosperi (INRA, Montpellier) and Fang Xie (SIBS, Shanghai) for providing seeds and plasmids, Magdalena Bezanilla (Dartmouth College) for the script for analysis of actin dynamics, and Rene Geurts (Wageningen Uni-versity) for providing a fluorescently labeled strain of S. meliloti. We would like to thank Philip Carella for critical reading; Diego H. Sanchez for helpful dis-cussions; and Annika Luebbe, Heena Yadav, and Bettina Hause for their tech-nical support and scientific discussions. This work was funded by the Gatsby Charitable Foundation (GAT3395/GLD), by the European Research Council (ERC-2014-STG, H2020, and 637537), and by the Royal Society (UF110073 and UF160413). T.R. was funded by the European Research Council (FP7-PEOPLE-2013-IEF, FP7, and 624398). S.B. was funded by the University of California, Los Angeles (403976-SB-69313), Gatsby Charitable Foundation (GAT3396/PR4), and Biotechnology and Biological Sciences Research Coun-cil (BB.L002884.1). J.L.K. was funded by the George and Lillian Schiff Founda-tion. D.R., E.-P.J., F.D., and F.d.C.-N. were funded by TULIP (no. ANR-10-LABX-41). V.C. is a recipient of a Thailand Research Fund grant
Figure 7.P. palmivora Infection of Chloroform-Treated API and api Plants
(A) Epifluorescence microscopy of chloroform-treated API and api seedlings 20 hpi after inoculation with P. palmivora LILI-td. Arrowheads indicate intra-radical growing hyphae; scale bars, 100mm.
(B) Average length of P. palmivora LILI-td intraradical hyphae per seedling (error bars represent SD; seedlings analyzed: n = 14 per genotype; t test: **p < 0.01).
(MRG6080235) and also supported by Faculty of Science, Mahidol University, Thailand.
AUTHOR CONTRIBUTIONS
A.G., T.R., T.A.T., J.L.K., D.R., F.D., S.B., and S.S. designed experiments. A.G., T.R., T.A.T., J.T., A.C., J.L.K., H.T., D.R., R.T., and S.B. performed exper-iments. A.G., T.R., T.A.T., J.L.K., H.T., V.C., D.R., S.B., and S.S. analyzed data. E.-P.J., F.D., and F.d.C.-N. provided materials. A.G., T.R., T.A.T., F.d.C.-N., S.B., and S.S. wrote the paper.
DECLARATION OF INTERESTS
The authors declare no competing interests. Received: May 28, 2020
Revised: July 8, 2020 Accepted: August 4, 2020 Published: September 3, 2020
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STAR
+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies
LM5 Rat monoclonal [48] N/A LM6 Rat monoclonal [49] N/A LM11 Rat monoclonal [50] N/A LM15 Rat monoclonal [51] N/A LM16 Rat monoclonal [52] N/A LM19 Rat monoclonal [52] N/A LM20 Rat monoclonal [52] N/A LM23 Rat monoclonal [53] N/A LM24 Rat monoclonal [53] N/A LM25 Rat monoclonal [53] N/A LM26 Rat monoclonal [44] N/A Rat IgG (whole molecule)-Peroxidase antibody
produced in rabbit
Sigma-Aldrich Cat# A9542; RRID:AB_258456 Rat IgG (whole molecule)-FITC antibody
produced in rabbit
Sigma-Aldrich Cat# F1763; RRID:AB_259443 Bacterial and Virus Strains
Agrobacterium rhizogenes arqua 1 Lab stocks N/A
Agrobacterium rhizogenes AR1193 Lab stocks N/A
E. coli TOP10 Chemically Competent C404010 N/A
Sinorhizobium meliloti 2011 [54] N/A
Chemicals, Peptides, and Recombinant Proteins
Brefeldin A Sigma-Aldrich Cat# B6542 m-maleimidobenzoyl-N-hydroxylsuccinimide ester Thermo Scientific Cat# 22311 Deposited Data
Microarray data http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE65903
GSE65903 Sequencing data http://www.ddbj.nig.ac.jp DRA003037 Sequencing data https://www.ncbi.nlm.nih.gov/sra SRR8468999 Sequencing data Bioproject PRJNA516327 Experimental Models: Organisms/Strains
Medicago truncatula Jemalong A17 Lab stocks N/A
M. truncatula Jemalong A17 api EMS mutant line [37] N/A
Lotus japonicus Gifu B-129 [16] N/A
L. japonicus scarn-1 EMS mutant line [16] N/A
Phytophthora palmivora AJ-td Lab stocks N/A
Phytophthora palmivora LILI-td Lab stocks N/A
Phytophthora palmivora LILI-YKDel Lab stocks N/A Oligonucleotides
Primers for plasmid construction seeTable S1 Eurofins N/A Recombinant DNA
pUB-GW-GFP-pUb:SCARN [16] N/A pUB-GW-GFP-pEp:SCARN [16] N/A
pUC57-API GENEWIZ N/A
pENTR:pAPI This paper N/A
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sebastian Schornack (sebastian.schornack@slcu.cam.ac.uk). All unique strains and plasmids used for this paper are available from the Lead Contact with a completed Materials Transfer Agreement.
EXPERIMENTAL MODEL AND SUBJECT DETAILS Plant materials and microbial strains
Medicago truncatula Jemalong A17 wild-type (referred as API) seeds were kindly provided by Dr. Jean-Marie Prosperi
(INRA-Mont-pellier). The api mutant used in this study is derived from the M. truncatula Jemalong A17 reference line after EMS mutagenesis and two backcrosses and displayed similar germination rate and seedling morphology as wild-type [37]. Medicago seed sterilization, germination and growth conditions have been described previously [10]. Agrobacterium rhizogenes-based root transformation of Medicago was performed according to Limpens et al. [57]. For the nodulation assay Medicago roots were inoculated with GFP-ex-pressing Sinorhizobium meliloti 2011 [54].
To allow tracking of the oomycete development in planta fluorescently labeled Phytophthora palmivora AJ-td (derived from acces-sion P6390) and LILI-td (derived from accesacces-sion P16830) carrying a pTOR:TdTomato vector or LILI-YKDel carrying a pTOR:CALYFP-KDEL were used for the infection assays. P. palmivora isolates were cultivated on V8 vegetable juice agar plates and used to infect
M. truncatula plants and to phenotype them as described previously [10]. Briefly, zoospores were released in cold water. The spore concentration was adjusted to 5x104spores ml–1. 10ml droplets of P. palmivora AJ-td or LILI-td spores were placed at the root tip of
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER pENTR:pHAPI1 This paper N/A pENTR:pHAPI2 This paper N/A pENTR:AtSCAR2 This paper N/A pKGW-MGW-pAPI:API This paper N/A pKGW-MGW-pUb:API This paper N/A pKGW-MGW-pUb:AtSCAR2 This paper N/A pK7GWIWG2-35S:hpAPI This paper N/A pKGW-GGRR-pHAPI1:GUS This paper N/A pKGW-GGRR-pHAPI2:GUS This paper N/A pKGW-MGW-pAPI:NLS:mTFP This paper N/A pKGW-MGW-pAPI:GUS This paper N/A pCAMBIA1300-35S:YFP-ABD2 This paper N/A pK7WGF2-pUb:SPpr1-GFP [43] N/A pKGW-MGW-pUb:PIP1-GFP This paper N/A Software and Algorithms
MATLAB function corr2 [55] N/A Actin_difference_maps This paper N/A Actin_skewness-and-occupancy This paper N/A Microsoft Excel Microsoft Office N/A AxioVision SE64 Rel. 4.9.1 N/A N/A RStudio Version 1.0.44 https://www.rsudio.com N/A R 3.0.2 https://www.r-project.org/ N/A Mega6 https://www.megasoftware.net/ N/A AGCC Scan Control Software http://www.affymetrix.com/ N/A GeneSpring GX 12.6.1 https://www.agilent.com/ N/A MAPMAN BINs https://mapman.gabipd.org/ N/A BWA software http://bio-bwa.sourceforge.net/ N/A Coval software https://omictools.com/coval-tool N/A MATLAB https://www.mathworks.com/ N/A
M. truncatula seedlings on 0.8% agarose plates. In each infection experiment a single application of zoospores was carried out. The same protocol was used for chloroform-treated seedling infection and xyloglucan supplemented infection. For chloroform treatment 1-day-old seedlings were soaked for 10 min in chloroform, rinsed several times with sterile water and infected on 0.8% agarose plates. High purity xyloglucan (Megazyme) was used to prepare a 50mM stock solution in deionized warm water. To obtain a working concentration of xyloglucan the necessary amount of stock solution was added directly to the spore suspension. Spore motility was checked microscopically before infection.
METHOD DETAILS
Genetic mapping and SNPs analysis
Previous work indicated that the api root hair and symbiotic phenotype are caused by a single recessive mutation, and the API locus was mapped to a 2.8 cM interval on the upper arm of M. truncatula linkage group 4 between markers MTIC331 and McSSR1 [37]. For finer mapping of the locus we screened 193 individuals of the F2 progeny from a cross between the M. truncatula api mutant (back-crossed twice in the A17 genetic background) and the M. truncatula wild-type line F83005.5. We identified 8 lines with recombination events in the above-defined interval between markers MTIC331 and EPJ005 and tested their symbiotic phenotype in F2 and F3 gen-erations. Using 6 additional molecular markers located between MTIC331 and EPJ005 based on M. truncatula genome sequence, we were able to position the API locus to an interval of about 550 kb (Figure S2A). To identify the api mutation we sequenced the api mutant (accession SRR8468999, Bioproject: PRJNA516327) and in parallel employed a comparative genome sequencing strategy.
API and api plant genomic DNA was extracted using DNeasy Plant Mini Kit (QIAGEN). The libraries for whole genome sequencing
were prepared with TruSeq DNA LT Sample Prep Kit (Illumina) and subjected to 100 bp paired-end sequencing by Illumina Hi-seq2500. Raw data can be found onhttp://www.ddbj.nig.ac.jp/using accession DRA003037. The sequence reads in which more than 10% of sequenced nucleotides had a phred quality score of less than 30 were excluded from the subsequent analysis. For de-tecting SNPs of api, we first constructed a ‘‘reference sequence’’ of API by replacing the nucleotides of the publicly available A17 M.
truncatula reference genome (ftp://ftp.jcvi.org/pub/data/m_truncatula/Mt4.0/Assembly/JCVI.Medtr.v4.20130313.fasta) with those of API as described previously [58]. A total of 122,523 SNP positions were substituted. The sequence reads from api were aligned to the developed API reference sequence using BWA software [59]. Alignment files were subjected to filtering using Coval software [60] and calculating the SNP-index for all SNP positions using the MutMap pipeline [61]. Finally the SNP positions having a sequence depth > 5 and showing an SNP-index > 0.9 were extracted as homozygous SNPs of api. Presence of the api causal mutation was confirmed via Sanger sequencing service at Source Bioscience (http://www.sourcebioscience.com/).
Design of constructs and cloning
Coding sequences of API (Medtr4g013235/MtrunA17_Chr4g0004861) were synthesized in pUC57 vector by GENEWIZ, Inc. Coding sequences of AtSCAR2 (AT2G38440) and PIP1 (Medtr8g098375/MtrunA17_Chr8g0386961) were amplified from Arabidopsis
thali-ana (Columbia-0 ecotype) and M. truncatula A17 cDNA, respectively. 2kb of 50 regulatory sequences of API, HAPI1 (Medtr7g071440/MtrunA17_Chr7g0244031) and HAPI2 (Medtr8g086300/MtrunA17_Chr8g0379031) were amplified from
M. truncatula A17 genomic DNA. PCR reactions were performed using Phusion DNA polymerase (New England Biolab Inc., UK)
and primers listed inTable S1. Amplicons were introduced into pENTR (D-TOPO Cloning Kit, Thermo Fisher Scientific) and used as an entry vectors. To generate complementation constructs entry vectors containing clones of API and AtSCAR2 were re-combined with pENTR:prAtUBQ3 into pKGW-MGW destination vector [62] using LR Clonase Plus (Thermo Fisher Scientific). To generate the PIP1-GFP reporter construct, the PIP1 entry vector was recombined into a pK7FWG2 destination vector. pENTR clones of promoters were recombined into a pKGW-GGRR destination vector, creating promoter GUS or mTFP fusions using LR Clonase Plus (Thermo Fisher Scientific).
Microscopy
For imaging of P. palmivora colonization in infected API and api root sections excised root tissues were mounted in water and covered by coverslips, using a Leica TCS SP8 confocal microscope with emission/excitation settings 561/570-600 nm for the P. palmivora AJ-td and 514/520-550 nm for the P. palmivora LILI-YKDel strain. Images represent stacks of 20 to 25 1-mm slices in maximum intensity projections merged with inverted transillumination images to outline cells. A line averaging of 4 was applied to reduce noise to signal ratio. Epifluorescence microscopy and sporangia counting were carried out using a Fluorescent Stereo Microscope Leica M165 FC equipped with a DFC310FX camera. The DSR filter (10447412) was used to detect the tdTomato produced by P. palmivora LILI-td or AJ-td. The same settings were used to screen for transgenic hairy roots of M. truncatula expressing monomeric DsRED. Light mi-croscopy imaging and infection scoring were performed using Zeiss Axioimager M2 microscope with a 64 MP color camera. Pictures were processed with ImageJ software v1.46 including Fiji plugins and AxioVision Microscopy Software (Zeiss).
Promoter GUS assay
Medicago transgenic roots were collected and washed twice in 0.1 M sodium phosphate buffer, pH 7.2, incubated in GUS buffer (100 mM sodium phosphate pH 7.0, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6and 2 mM 5-bromo-4-chloro-3-indoxyl-b-d- glucuronic
acid, X-gluc) under vacuum at room temperature for 30 min to allow the buffer to replace air in the tissue, incubated at 37C for 2-6 h to enable the enzymatic reaction, and analyzed using the Microscope Leica M165 FC and Zeiss Axioimager M2 microscope.
